Dissertation / PhD Thesis

FZJ-2014-02842

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Metabolic engineering of $\textit{Corynebacterium glutamicum}$ for production of L-leucine and 2-ketoisocaproate

Vogt, M. (Corresponding Author)FZJ*

2014
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-968-3

Please use a persistent id in citations: http://hdl.handle.net/2128/7855

Abstract: Due to the depletion of fossil energy sources, there is an increasing demand for alternative sustainable production processes utilizing renewable resources. Biotechnological approaches using microorganisms such as $\textit{Corynebacterium glutamicum}$ as biocatalysts play an important role for the production of beneficial substances. These include the three branched-chain amino acids L-valine, L-isoleucine, and L-leucine, as well as their respective keto acid precursors, which have diverse commercial applications in food, feed, and pharmaceutical industry. In this work, metabolic engineering of the $\textit{C. glutamicum}$ wild type was employed to develop efficient strains for the production of L-leucine and 2-ketoisocaproate. The key-player enzyme in L-leucine biosynthesis is the $\textit{leuA}$-encoded 2-isopropylmalatesynthase which is feedback-inhibited by low L-leucine concentrations with a K$_{i}$ of 0.4 mM. A feedback-resistant variant of the 2-isopropylmalate synthase was identified and characterized biochemically in the available weak L-leucine producer B018, which had been obtained by random mutagenesis and screening. The respective gene $\textit{leuA}$_B018, devoid of the attenuator region and under control of a strong promoter, was integrated in up to three copies into the genome of $\textit{C. glutamicum}$ wild type and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved I) deletion of the gene $\textit{ltbR}$ encoding the repressor LtbR to increase expression of genes $\textit{leuBCD}$, II) deletion of the gene $\textit{iolR}$ encoding the transcriptional regulator IolR to increaseglucose uptake, III) reduction of citrate synthase activity to increase precursor supply, and IV) introduction of a modified $\textit{ilvN}$ gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in shake flask and bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of approximately 24 g l$^{-1}$. The maximal molar product yield and volumetric productivity were 0.30 mol per mol glucose and 4.3 mmol l$^{-1}$ h$^{-1}$, respectively. Moreover, the achieved values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application. Based on the described L-leucine producers, strains for the production of the keto acid 2-ketoisocaproate, the direct precursor of L-leucine, were constructed and characterized as well. This was achieved by deletion of the gene encoding the transaminase llvE or replacement of the ATG start codon of $\textit{ilvE}$ by GTG start codon. The resulting strains accumulated up to 6.1 g l$^{-1}$ 2-ketoisocaproate in shake flask cultivations, with a molar product yield of 0.20 mol per mol glucose and a volumetric productivity of 1.4 mmol l$^{-1}$ h$^{-1}$.

Keyword(s): Dissertation

Note: Biotechnologie 1
Note: Heinrich-Heine-Universität Düsseldorf, Diss., 2014

---

Contributing Institute(s):

1. Biotechnologie (IBG-1)

Research Program(s):

1. 899 - ohne Topic (POF2-899) (POF2-899)

Appears in the scientific report 2014

---

Database coverage:

---